Ultrasound Visualization And Targeted Degradation Of PDL1 In Tumor Based On Engineered Extracellular Vesicles

Background:Programmed cell death 1 ligand 1（PDL1）immune checkpoint inhibitors hold promise for cancer therapy.However,PDL1 is widely expressed on tumor and stromal cells and with extensive heterogeneity.Evaluation of PDL1 expression in vivo is of great significance for scientifically guiding to immunotherapy.Ultrasound molecular imaging is a potential way to achieve dynamic monitoring of PDL1,but traditional ultrasound contrast agents have difficulties to pass through tumor blood vessels or have poor echogenic sensitivity.As a natural membrane-derived carrier,the extracellular vehicles（EVs）have the advantages of good stability and high biocompatibility.EVs are also easy to be engineered to increase their targeting through a variety of surface modification strategies.After modification,they can target different molecules in tumor tissue,which makes them become the potential ideal vector of ultrasound contrast agent.Aims:We constructed an ultrasound contrast agent targeting PDL1,denoted as Gp-EV^tPD1.With the help of PDL1-targeted contrast agent,the expression of PDL1 was monitored in real time and accurately evaluated by ultrasound molecular imaging before or during treatment,and investigated the therapeutic effect of Gp-EV^tPD1 by mediating the endocytosis and degradation of PDL1.Methods:1.Truncated PD1（tPD1）was fused with EV-abundant transmembrane protein prostaglandin F2 receptor negative regulator（PTGFRN）and displayed on the surface of EVs by genetic engineering modification,and performed verification of characterization and targeting effect.2.The engineered EVs are then encapsulated with Ca（HCO₃）₂ via electroporation,which would recognize PDL1 highly expressed cells and produce gas in the endosomes and lysosomes.For the verification of the ultrasound imaging effect of Gp-EV^tPD1,agarose phantom was used to simulate the tissue echogenicity in vitro.After the Gp-EV^tPD1 was co incubated with the recipient cells for 0,2,4 and 6 hours,the ultrasound examination was performed;In vivo,tumor bearing mice were injected with Gp-EV^tPD1 via tail veins,and ultrasound examination was performed before injection and 0,2,4,and 6 hours after injection.3.tPD1 on the EV binds PDL1 and triggers the PDL1 endocytosis and degradation in endosomes/lysosomes in a sequential manner,and thus boosts the anti-tumor immunity of cytotoxic T cells.For the verification of anti-tumor immunotherapy effect,tumor bearing mice were treated by tail vein injection every two days for two weeks.After treatment,the anti-tumor therapeutic effect was observed by measuring the tumor size and the survival rate of tumor bearing mice,and the tumor immune status was observed by measuring the infiltration of CD8⁺T cells and the expression level of related cytokines in the tumor by flow cytometry.Results:1.After engineering modification,the particle size and characterization of Gp-EV^tPD1were not significantly changed compared with unmodified EVs,had targeting effect on tumor tissues which highly expressed PDL1.2.In vitro,after the incubation of Gp-EV^tPD1 with recipient cells,the ultrasonic echogenicity was significantly enhanced compared with the control group;In vivo,as a contrast agent,Gp-EV^tPD1 significantly enhanced the echogenicity of PDL1 expressing tumor tissue.The signal reached the maximum at 4 hours,and the echogenicity intensity was correlated with the PDL1 expression level.3.After Gp-EV^tPD1treatment,the expression of PDL1 in tumors decreased,the level of IFNγand TNFαand proportion of CD8⁺T cells increased.When compared with the control group,a significant decrease in tumor volume was observed.Conclusions:Using Gp-EV^tPD1 as ultrasound contrast agent can realize the visualization and real-time evaluation of PDL1 expression level through ultrasound imaging,which can be used to guide the immunotherapy and reduce drug resistance and side effects.At the same time,Gp-EV^tPD1,as a new PDL1 blocker,can relieve tumor immunosuppression while visualizing PDL1 expression,play the role of ultrasound theranostic,and shade a light for tumor immunotherapy.